Journal: Journal of Neurochemistry

Article Title: Modulation of pTau181 by Glypican‐1‐Derived Heparan Sulfate in Human Neural Progenitor Cells and ApoE4 ‐Expressing Induced Neurons

doi: 10.1111/jnc.70162

Figure Lengend Snippet: Processing of amyloid precursor protein (APP) and glypican‐1 (GPC1) in late endosomes (top) and phosphorylation of cytosolic tau (bottom). β‐NTF and β‐CTF, N‐terminal and C‐terminal APP fragments, respectively; Aβ, amyloid beta peptides; HS‐anMan, heparan sulfate with reducing terminal anhydromannose (blue pentagon); SNO, nitrosothiol; SH, thiol; N =N, molecular nitrogen generated from SNO in the redox reaction; Asc, ascorbate; GPI, glycosyl‐phophatidyl‐inositol; Red squares in tau, microtubule‐binding region; P , phosphate groups. GPC1 is the major, if not exclusive, source of HS‐anMan in human neural progenitor cells and neural stem cells (Cheng et al. , ).

Article Snippet: For dissociation and re‐plating, Accutase (Fisher Scientific Cat. # 11599686) was used as described in detail previously (Cheng et al. ). iPSC‐derived human neuronal progenitor cells (NPC, ATCC, Cat. # ACS‐5003) were plated in CellMatrix gel‐coated plates (Growth kit ACS‐3003 for NPC expansion).

Techniques: Phospho-proteomics, Generated, Binding Assay

Journal: Fluids and Barriers of the CNS

Article Title: Translational biomarkers of hypoxic brain injury uncovered in CSF secreting human choroid plexus organoids

doi: 10.1186/s12987-025-00731-z

Figure Lengend Snippet: Generation of human cerebral and ChP organoids with fluid-filled cavities. ( A ) Protocol timeline with images of ChP and COs over time. The black arrow indicates emerging ChP epithelium at day 9 and the arrowhead shows a later fluid-filled compartment. “d” indicates “day”, scale bar 150 μm. ( B ) Comparison of ChP organoid and COs (day 9 and day 120). The black arrow indicates ChP epithelium and the arrowhead fluid-filled compartment. Scale bar 150 μm. ( C ) Immunofluorescence of ChP (GMB7-1 cell line) and cerebral (BXS0115, MAA3) organoids. Identification of different cell types present in these organoids: mature neuron (MAP2 in red and NEUN in green), astrocytes (GFAP in green and GLAST in red), radial glia (PAX6 in green) and neural progenitor (SOX2 in red). Scale bar 150 μm, 10X objective for marker PAX6/MAP2; NEUN/SOX2 and 20X objective for GFAP/MAP2 and GLAST/NEUN. ( D ) Immunofluorescence of ChP (GMB7-1 cell line) organoids with tight junction markers CLDN5, ZO1 and MDR-1. Scale bar 50 μm, objective 40X and 63X. ( E ) Heatmap with different proteins expression between MAA3, BXS0115 and GMB7-1 cell line organoids explaining potential differentiation of GMB7-1 cell line into ChP organoid

Article Snippet: ATCC cell line BXS0115 human induced pluripotent stem cells were reprogrammed from parental cell line CD34- bone marrow cells using Sendai virus expression of oct4, klf4 and Myc genes.

Techniques: Comparison, Immunofluorescence, Marker, Expressing

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: Persistent doxorubicin-induced cardiomyocyte damage and apoptosis despite dexrazoxane interventions (A) Cardiomyocytes were treated with 1 µM doxorubicin (DOX) for 24 h and subsequently cultured in a fresh medium without DOX for an additional 24 h. Cell viability was assessed using a CCK-8 assay. The results demonstrate a significant reduction in cell viability following DOX treatment (* p < 0.05 vs. 0 µM), which persists even after the removal of the drug, indicating irreversible viability loss. (B) Treatment with dexrazoxane (DRZ) at 50 µM restored cell viability to levels comparable to control, whereas concentrations above 100 µM appeared to have diminished protective effects. (C) Apoptotic cells were detected using annexin V staining. Representative images show increased annexin V-positive cardiomyocytes after 24 h of DOX treatment and continued apoptosis 24 h post-drug removal. Quantitative analysis of annexin V-positive cells confirmed significant apoptosis induced by DOX, which remains elevated after the removal of the drug. (D) Western blotting of cleaved caspase-3 in cardiomyocyte lysates. Cropped gels and blots are displayed, with full-length blots provided in Supplementary Fig. . The results demonstrate increased levels of cleaved caspase-3 following DOX treatment, which remain elevated even after drug removal, suggesting persistent activation of the apoptotic pathway. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Cell Culture, CCK-8 Assay, Control, Staining, Western Blot, Activation Assay

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: ASCs decreased doxorubicin-induced apoptosis and caspase-3 activation through paracrine effects in a Transwell coculture system (A) Differentiation of ASCs into adipocytes and osteocytes was confirmed using Oil Red O staining and Alizarin Red staining. Scale bar, 100 μm. (B) Comparing surface markers of ASCs in MesenPro medium and Complete Claycomb medium. (C) Schematic diagram of the Transwell coculture experimental timeline. HL-1 cardiomyocytes were seeded into the lower wells of a 6-well plate, and ASCs were seeded into the upper Transwell inserts. Both cell types were maintained in a Complete Claycomb medium (containing 10% FBS) throughout the experiment. After a 4-hour coculture adaptation period, DOX was added to induce cardiotoxicity. ASCs were retained in the upper chamber during DOX exposure to maintain continuous paracrine signaling and simulate coexposure conditions. Scale bar, 100 μm. (D) Caspase-3 activation was assessed in cardiomyocytes treated with 1 µM DOX alone or in coculture with ASCs using Western blotting. Cropped gels and blots are displayed, with full-length blots provided in Supplementary Fig. . (E) Quantitative analysis of annexin V-positive cells and cleaved caspase-3 bands normalized to GAPDH confirmed the statistically significant reduction in apoptosis and caspase-3 activation in the presence of ASCs compared to DOX treatment alone. * p < 0.05; ** p < 0.01; *** p < 0.005

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Activation Assay, Staining, Western Blot

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: CM attenuated DOX-induced apoptosis in cardiomyocytes (A) The schematic illustrates the timeline of cell seeding, coculture, and treatment phases. CM was prepared by culturing ASCs in DMEM-HG supplemented with 10% standard FBS for 72 h, followed by filtration. All DOX-only and control groups were cultured in DMEM-HG medium containing 10% FBS to confirm that CM-specific effects were not due to FBS. (B) Adipose-derived stem cells (ASCs) secrete a range of pro-angiogenic and cardioprotective factors, including hepatocyte growth factor (HGF), granulocyte colony–stimulating factor (G-CSF), insulin-like growth factor 1 (IGF-I), platelet-derived growth factor (PDGF), placental growth factor (PIGF), vascular endothelial growth factor (VEGF), stem cell factor (SCF), epidermal growth factor (EGF), transforming growth factor (TGF-β1), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (Ang1), interleukin (IL)-6, and thrombopoietin (TPO). (C) HL-1 cardiomyocytes were pretreated with CM derived from either mitomycin C-treated ASCs (CM MMC−treated ASC ) or untreated ASCs (CM proliferative ASC ) for 4 h prior to doxorubicin (DOX, 1 µM) exposure, and CM was replenished during the 24-hour DOX treatment. Cell viability was assessed using the CCK-8 assay. (D) The impacts of ASC-derived CM on DIC, as determined by annexin V and propidium iodide (PI) staining, highlight its role in reducing cardiomyocyte apoptosis. * p < 0.05; *** p < 0.005; **** p < 0.0001

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Filtration, Control, Cell Culture, Derivative Assay, CCK-8 Assay, Staining

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: EV fulfilled with Minimal Information for Studies of Extracellular Vesicles 2023 guidelines interacts with cardiomyocytes (A) Schematic representation of the isolation of extracellular vesicles (EVs) using the combination of qEV size exclusion columns (SEC) and an automatic fraction collector, followed by characterization and tracking of EVs in HL-1 cardiomyocytes. The first six fractions (F1-F6) after the buffer were collected, with each fraction being 0.7 mL. (B) Simultaneous measurement of size and concentration using tunable resistive pulse sensing (TRPS). (C) Statistical analysis of particle diameters. (D) Concurrent measurement of both size and zeta potential via TRPS. (E) Transmission electron microscopy (TEM) images. (F) Detection of internal markers; cropped gels and blots are shown, with full-length blots available in Supplementary Fig. . (G) Profiling of the exosomal marker CD63 within EVs and Dulbecco’s Phosphate-Buffered Saline (DPBS). (H) Profiles of growth factors present in EVs. (I) Interaction and uptake of EVs by HL-1 cardiomyocytes, emphasizing their proximity to mitochondria. Two co-staining protocols were utilized: the first employed Hoechst for nuclei, PlasMem for cell membranes, and ExoSparkler for exosomal membranes; the second used Hoechst for nuclei, MitoBright for mitochondria, and ExoSparkler for exosomal membranes. Colocalization of EVs with mitochondrial markers suggests their involvement in modulating mitochondrial function and oxidative stress in cardiomyocytes, providing insights into potential therapeutic mechanisms against DIC. Scale bar, 100 μm. Magnified view of the white-boxed region. Scale bar, 10 μm. HCI = High-Content Imaging System. SEC = size exclusion chromatography

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Isolation, Concentration Assay, Tunable Resistive Pulse Sensing, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Marker, Saline, Staining, Imaging, Size-exclusion Chromatography

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: EV restored the viability of HL-1 cardiomyocytes post-DOX exposure (A) Interaction and uptake of extracellular vesicles (EVs) by HL-1 cardiomyocytes 1 h after DOX exposure, compared to control cells, demonstrating initial EV internalization. (B) Interaction and uptake of EVs by HL-1 cardiomyocytes 20 h after DOX exposure, compared to control cells, indicating sustained EV presence and cellular uptake over time. (C) Impacts of EVs and their vehicle, PBS, on control cells and DOX-treated cells, providing a baseline for EV interaction in the absence of DOX-induced stress. (D) Effects of EV treatment on cardiomyocyte survival at the end of the 24-hour DOX exposure period and 24 h after DOX removal, highlighting the sustained cardioprotective effects of EVs in mitigating DOX-induced cytotoxicity. HCI = High-Content Imaging (HCI) System. * p < 0.05; ** p < 0.01; **** p < 0.0001. Scale bar, 100 μm. Magnified view of the white-boxed region. Scale bar, 10 μm

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Control, Imaging

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: Clusterin played a critical role in CM and EVs against DOX-induced apoptosis (A) Venn diagram of differentially expressed genes (DEGs) across three experimental groups. A total of 621 genes uniquely modulated by CM in response to DIC are highlighted within the green circle. The accompanying volcano plot illustrates the statistical significance and magnitude of changes in the expression of these 621 genes, facilitating the rapid identification of significant DEGs. (B) Heat maps of protein-coding DEGs with TPM values above 5 in the CM-treated group are clustered hierarchically to reveal expression patterns across samples. (C) KEGG pathway analysis highlights representative pathways featuring the top 7 gene ratios. Gene ratio (x-axis) is the percentage of the number of genes present in this GO term over the total number of genes in this category. (D) The top 5 terms from the functional analysis of biological processes based on false discovery rate (FDR) in the CM-treated group are presented. (E) The quantification of clusterin (CLU) levels in HL-1 cardiomyocyte lysates and supernatants shows an upregulation following CM or EV treatment. (F) A schematic representation explains the siRNA-mediated knockdown of clusterin, outlining the experimental workflow, including quantification of clusterin in HL-1 cardiomyocyte lysates (intracellular CLU) and supernatant (secreted CLU) post-siRNA treatment, confirming effective knockdown. (G) Metabolic activity assessments reveal comparable protective effects of CM and EVs, as demonstrated by CCK-8 assays. (H) A representative Western blot shows caspase-3 activation, illustrating the protective effect of CM and EV treatment. Cropped gels and blots are shown, with the full-length blots available in Supplementary Fig. 6. Densitometric analysis of Western blot data provides statistical validation. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001. si-CLU = siRNA targeting clusterin; si-Ctrl = negative control siRNA; TR = Lipofectamine RNAiMAX Transfection Reagent. None = no siRNA or TR treatment

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Expressing, Functional Assay, Knockdown, Activity Assay, CCK-8 Assay, Western Blot, Activation Assay, Biomarker Discovery, Negative Control, Transfection

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: CM activated AKT and reduced mitochondrial superoxide production (A) Representative Western blot images illustrating the levels of caspase-3 activation alongside the phosphorylation of AKT, BAD, and GSK3β in response to CM treatment, highlighting the regulatory effects of CM on AKT-BAD and AKT-GSK3β signaling. Cropped gels and blots are shown, with full-length blots available in Supplementary Fig. . Densitometric analysis of the Western blot results provides quantitative validation of the observed protein expression changes. (B) Representative high-content images (HCI) show co-staining for Hoechst (nuclei), MitoBright (mitochondria), and mtSOX (mitochondrial superoxide), along with merged panels that illustrate cellular localization and oxidative stress response. Enlarged images from the boxed regions highlight the colocalization of mitochondria and oxidative stress markers, providing deeper insight into the intracellular effects of CM treatment. Scale bar, 100 μm. Magnified view of the white-boxed region. Scale bar, 10 μm. (C) A representative histogram of flow cytometry analysis displays the fluorescence of mtSOX superoxide. (D) Quantitative analysis of mtSOX fluorescence intensity is conducted using both HCI and flow cytometry to assess oxidative stress levels in cardiomyocytes across different treatment conditions. (E) Oxygen consumption rate (OCR) measurement by using the Seahorse XFe96 extracellular flux analyzer at baseline and after the addition of oligomycin, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone + antimycin A. Mitochondrial function, focusing on basal and maximal respiration in HL-1 cardiomyocytes pretreated with DRZ, CM, or EV, was compared to groups treated only with DOX. (F) Control experiments verified that treatment with LY294002 alone, ranging from 5 to 20 µM, did not induce significant cytotoxicity. (G) HL-1 cells were pretreated with LY294002 (20 µM) for 2 h before the addition of CM and/or DOX. After pretreatment, cells were exposed to DOX (1 µM) and CM for 24 h. Caspase-3 cleavage was subsequently assessed by Western blotting to evaluate apoptosis. The experimental groups included: (1) Control (Ctrl), (2) DOX-only, (3) DOX + CM, (4) DOX + CM + LY294002, and Ctrl + LY294002. * p < 0.05; ** p < 0.01; *** p < 0.005; **** p < 0.0001

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Biomarker Discovery, Expressing, Staining, Flow Cytometry, Fluorescence, Control

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: EVs restored metabolic activity and clusterin expression in induced pluripotent stem cell-derived human cardiomyocytes exposed to DOX treatment (A) Metabolic activity of cardiomyocytes was evaluated using the CCK-8 assay. (B) Spontaneous beating rates of human cardiomyocytes were recorded using an inverted phase-contrast microscope equipped with a high-resolution digital camera. (C) Clusterin expression levels were quantified via ELISA. * p < 0.05; ** p < 0.01; **** p < 0.0001

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques: Activity Assay, Expressing, Derivative Assay, CCK-8 Assay, Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Biology Direct

Article Title: Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

doi: 10.1186/s13062-025-00664-5

Figure Lengend Snippet: Schematic representation of the EV-enriched secretome from ASCs, which modulates mitochondrial ROS, reduces apoptosis, and increases clusterin in cardiomyocytes treated with DOX

Article Snippet: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes were cultured following the manufacturer’s instructions (Cell Applications, USA).

Techniques:

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Formation of mouse primary cardiomyocyte clusters in agarose-coated wells. ( a ) Schematic drawing of the conventional dish cultivation of cardiomyocytes. The dispersed cells were cultured on the bottom of a 35-mm non-agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells attached on the bottom of the 35-mm cultivation dish dispersedly. The cells started to beat 2–3 days after cultivation started. ( b ) A micrograph of dispersed cardiomyocytes in a 35-mm non-agarose-coated dish. ( c ) Schematic drawing of the cultivation of dispersed cells in a 35-mm agarose-coated dish. After spread of the 2.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5.0\times 10^{4}\, {\rm cells/mL}$$\end{document} 5.0 × 10 4 cells / mL isolated single cardiomyocytes, the cells dispersed on the bottom of the agarose layer in the agarose-coated 35-mm cultivation dish. Even after 2–3 days of cultivation, the cells remained isolated with a round shape, and no clusters formed on the bottom. ( d ) A micrograph of cardiomyocytes in an agarose-coated 35-mm cultivation dish. ( e ) Schematic drawing of the cultivation of dispersed cells in a 15.5-mm agarose-coated cultivation well (in a 24-well cultivation plate). After spread of the 1.0 mL of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$5\times 10^{4}\hbox { cells/mL}$$\end{document} 5 × 10 4 cells/mL isolated single cardiomyocytes, dispersed cells gathered and formed small clusters; finally, they gathered into a single large cluster in the 15.5-mm agarose-coated cultivation well. ( f ) A micrograph of a cardiomyocyte cluster in a 15.5-mm agarose-coated cultivation well.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Cell Culture, Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Micrographs of single cells and clusters of mouse primary and hES-derived cardiomyocytes. ( a ) Mouse primary cardiomyocytes (primary) in a 35-mm non-agarose-coated dish (single cell), ( b ) primary cells in a 24-well agarose-coated plate (cluster), ( c ) hES cardiomyocytes in a 35-mm non-agarose-coated dish (single cell), and ( d ) hES in a 24-well agarose-coated plate (cluster).

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Derivative Assay

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Analysis of interbeat interval (IBI) distribution of single and clustered mouse primary and hES cardiomyocytes. ( a )–( d ): Method of measuring interbeat interval (IBI) of single cardiomyocytes and clusters. Temporal change of luminance in the red square area for single cell ( a ) and cluster ( c ) caused by their beating was recorded, as shown in the time-course intensity profiles ( b ) and ( d ), respectively. IBIs of their beating were acquired from the time intervals between two neighboring peaks in the time-course intensity profiles. ( e ), ( f ): Distribution of IBIs of mouse primary cardiomyocytes. ( e ) The relationship between mean IBIs and fluctuations of beating [coefficient of variability (CV) of IBIs] of single isolated primary cardiomyocytes (blue open circles, n = 73) and primary clusters (red filled triangles, n = 6). ( f ) A histogram of all plots in ( e ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean value and standard deviation (SD) of single-cardiomyocyte IBIs, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters. ( g ), ( h ): Distribution of IBIs in hES cardiomyocytes. ( g ) The relationship between mean IBIs and CV of IBIs in single isolated hES cardiomyocytes (blue open circles, n = 125) and hES clusters (red filled triangles, n = 27). ( h ) A histogram of all plots in ( g ). The blue filled bars indicate the frequency of IBIs of single cardiomyocytes; the blue arrow and the error bar indicate the corresponding mean values and SD of single cardiomyocytes, respectively. The red filled bars indicate the frequency of IBIs of clusters; the red arrow and the error bar indicate the corresponding mean value and SD of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation, Standard Deviation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distribution of IBIs and fluctuation of IBI distribution of the hES cardiomyocyte clusters and their constituent cells. ( a )–( c ): Micrographs of hES cardiomyocyte clusters. ( d )–( f ): Distribution of IBIs and the CV of IBIs in the clusters ( a )–( c ) and isolated constituent cells from each cluster (n=50 from among re-cultivated \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$1.0\times 10^{3}\hbox { cells}$$\end{document} 1.0 × 10 3 cells ). These plots ( d )–( f ) correspond to each cluster ( a )–( c ). The red filled triangles indicate the cardiomyocyte clusters, and the blue open circles indicate constituent cardiomyocytes of each cluster. Each cluster was measured 2 days after the beating started. Single cardiomyocytes were isolated from each cluster by trypsinization. IBIs of single constituent cardiomyocytes were measured 3 days after their isolation. Median and 95% confidence interval of single cardiomyocytes were 0.971 s and 0.825–1.25 s ( d ), 1.19 s and 1.00–1.28 s ( e ), and 1.12 s and 0.844–1.22 s ( f ), respectively. ( g )–( i ): Histograms of IBIs of each cluster and its isolated constituent cells. The blue filled bars indicate the ratio of frequency for single constituent cardiomyocytes; the blue arrows and error bars indicate the mean IBIs and SDs, and the red arrows also indicate the mean IBIs of clusters.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques: Isolation

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Influence of trypsinization on interbeat intervals in dispersed individual hES cardiomyocytes. ( a ) Distribution of the IBIs and the CV of IBIs in single hES cardiomyocytes before and after trypsinization. The blue open circles indicate the single hES cardiomyocytes (n = 50) before trypsinization. The orange open circles indicate the single cardiomyocytes (n = 50) after trypsinization. ( b ) Histograms of IBIs of hES single cardiomyocytes before and after trypsinization. The blue filled bars indicate the mean IBIs of single cardiomyocytes before trypsinization; the blue arrow and error bar indicate their mean value and SD. The orange filled bars indicate the frequency of mean IBIs of trypsinized single cardiomyocytes; the orange arrow and error bar indicate their mean value and SD.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Scientific Reports

Article Title: Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

doi: 10.1038/s41598-021-91466-y

Figure Lengend Snippet: Distributions of interbeat intervals (IBIs) and fluctuations of the two hES cardiomyocyte clusters before and after their connection and after re-separation. ( a )–( e ): Micrographs of cardiomyocyte clusters. Micrographs of the large cluster ( a ) and small cluster ( b ) before contact. These clusters were measured when they had been cultivated for 7 days. The two hES cardiomyocyte clusters were connected ( c ). The measurement was performed 3 days after the two clusters contacted each other. Micrographs of the large cluster ( d ) and small cluster ( e ) after separation. The measurements were taken within 5 min of separation. ( f ): Distribution of IBIs and fluctuations of two clusters before contact, during contact, and after separation. Blue filled bar and error bar indicate the mean IBIs and SD of the large cluster. Green filled bar and error bar indicate the mean IBIs and SD of the small cluster.

Article Snippet: Human embryonic stem cell-derived cardiomyocytes (hES) (hES-CMCTM002, hES cell line SA002) were purchased from Cellectis (Gothenburg, Sweden) , .

Techniques:

Journal: Heart rhythm

Article Title: Disruption of protein quality control of the human ether-à-go-go related gene K + channel results in profound long QT syndrome

doi: 10.1016/j.hrthm.2021.10.005

Figure Lengend Snippet: Colocalization of hERG and RNF207 in guinea pig ventricular cardiomyocytes. A: Confocal images showing colocalization among α-actinin2, hERG K+ channel subunits, and RNF207. Scale bar = 10 μm. The right panels show the corresponding fluorescence intensity profiles perpendicular to the z lines. B: Proximity ligation assay (PLA) for α-actinin2, hERG K+ channel subunits, and RNF207. C: Quantification of PLA signals per cell area (puncta/μm2). n = 15, 11, 15, 10, 9, and 9 cells from left to right bars; *P < .05. D: Auto-ubiquitinylation assay for RNF207WT (lane 3, right) vs negative control (lane 1, left) and MDM2, a known E3 ubiquitin ligase (positive control, lane 2). Transfected HEK 293 cells were immunoprecipitated (IP) for RNF207-FLAG. An auto-ubiquitinylation assay was conducted on isolated protein, followed by SDS-PAGE and Western blot analysis (IB). Proteins were incubated in the presence of E1 and E2 ubiquitin enzymes, ubiquitin, and ATP. E: Ubiquitination assays. Ubiquitinated proteins were absent in the negative control (lane 1). The E3-ubiquitin band appeared for hERGT613M incubated with RNF207WT (lane 3), but not in the presence of RNF207G603fs (lane 5) or with hERGWT subunits (either with RNF207WT [lane 2] or with RNF207G603fs [lane 4]). F: Quantification of the data from panel E. n = 5 independent experiments for each group; *P < .05. Data shown are mean ± SEM. Analyses were performed using 1-way analysis of variance (ANOVA) with Brown-Forsythe post hoc analyses. AU = arbitraty units; HEK 293 = human embryonic kidney 293; hERG = human ether-à-go-go related gene; RNF207 = ring finger protein 207; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM = standard error of the mean; Ub = ubiquitin; WT = wild-type.

Article Snippet: We took advantage of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs, iCell, FUJIFILM Cellular Dynamics, Inc., Madison, WI) as a platform ( – ).

Techniques: Fluorescence, Proximity Ligation Assay, Negative Control, Positive Control, Transfection, Immunoprecipitation, Isolation, SDS Page, Western Blot, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Heart rhythm

Article Title: Disruption of protein quality control of the human ether-à-go-go related gene K + channel results in profound long QT syndrome

doi: 10.1016/j.hrthm.2021.10.005

Figure Lengend Snippet: Regulation of APDs of hiPSC-CMs by RNF207. A: Representative action potential recordings (iCell, Cellular Dynamics) in cells expressing hERGWT:RNF207WT (black trace), hERGWT:hERGT613M:RNF207WT (red trace), and hERGWT:hERGT613M:RNF207WT:RNF207G603fs (blue trace) as well as a nontransfected cell (gray trace). B–F: Summary data for action potential recordings in nontransfected cells (labeled “Non-TF”; gray bar) compared with hERGWT:RNF207WT (labeled “WT”; black bar), hERGWT:hERGT613M:RNF207WT (labeled “Rescue”; red bar), and hERGWT:hERGT613M:RNF207WT:RNF207G603fs (labeled “Mutant”; blue bar) at baseline (solid bars) vs 1 μM E-4031 (striped bars). Data are shown for average diastolic potential (panel B), peak action potential (panel C), action potential amplitude (panel D), action potential duration at 50% repolarization or APD50 (panel E), and action potential duration at 90% repolarization or APD90 (panel F). Data shown represents the average of 5 action potentials per cell, with n = 6–9 cells for baseline recordings and n = 3–5 cells for E-4031 recordings. *P<.05, **P<.01, §P<.001. Data shown are mean ± SEM. Analyses were performed using ANOVA with Tukey’s post hoc analyses. ANOVA = analysis of variance; APD = action potential duration; APD50 and APD90 = APD at 50% and 90% repolarization; hERG = human ether-à-go-go related gene; hiPSC-CM = human induced pluripotent stem cell-derived cardiomyocytes; non-TF = nontransfected cells; RNF207 = ring finger protein 207; SEM = standard error of the mean; WT = wild-type.

Article Snippet: We took advantage of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs, iCell, FUJIFILM Cellular Dynamics, Inc., Madison, WI) as a platform ( – ).

Techniques: Expressing, Labeling, Mutagenesis, Derivative Assay

Journal: Heart rhythm

Article Title: Disruption of protein quality control of the human ether-à-go-go related gene K + channel results in profound long QT syndrome

doi: 10.1016/j.hrthm.2021.10.005

Figure Lengend Snippet: Regulation of hERG currents by RNF207 in hiPSC-CMs and a schematic diagram of RNF207 interaction with hERG-encoded K+ channels in adult ventricular myocytes. A: Representative E-4031–sensitive currents recorded from hiPSC-CMs expressing hERGWT:RNF207WT (black traces), hERGWT:hERGT613M:RNF207WT (red traces), and hERGWT:hERGT613M:RNF207WT:RNF207G603fs (blue traces). B: Summary data of current density for the 3 groups of cells. n = 5–6. C: Summary data for voltage-dependent activation using the peak tail current density fitted using the Boltzmann function (see Online Supplemental Table 1). n = 5–6. In panel B, *P < .05 for hERGWT:RNF207WT compared with hERGWT:hERGT613M:RNF207WT:RNF207G603fs throughout positive voltages and was shown only at the end of the curve for clarity. In panel C, *P < .05 for hERGWT:RNF207WT and hERGWT:hERGT613M:RNF207WT compared with hERGWT:hERGT613M:RNF207WT:RNF207G603fs throughout positive voltages and was shown only at the end of the curves for clarity. Analyses were performed using ANOVA with Tukey’s post hoc analyses. D: Schematic diagram of RNF207 interaction with hERG-encoded K+ channels (Kv11.1) with trafficking and degradation pathways (generated using BioRender, Toronto, Canada). ANOVA 5 analysis of variance; hERG = human ether-à-go-go related gene; hiPSC-CM 5 human induced pluripotent stem cell-derived cardiomyocytes; I = current; RNF207 = ring finger protein 207; SEM = standard error of the mean; V = voltage; WT = wild-type.

Article Snippet: We took advantage of human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs, iCell, FUJIFILM Cellular Dynamics, Inc., Madison, WI) as a platform ( – ).

Techniques: Expressing, Activation Assay, Generated, Derivative Assay